1. Field of the Invention
The present invention relates to the field of engineered peptides, and to the field of peptides which bind to integrins, and, particularly to integrin binding as it relates to cell growth and development.
2. Related Art
Integrins are a family of extracellular matrix adhesion proteins that noncovalently associate into α and β heterodimers with distinct cellular and adhesive specificities (Hynes, 1992; Luscinskas and Lawler, 1994). Cell adhesion, mediated though integrin-protein interactions, is responsible for cell motility, survival, and differentiation. Each α and β subunit of the integrin receptor contributes to ligand binding and specificity.
Protein binding to many different cell surface integrins can be mediated through the short peptide motif Arg-Gly-Asp (RGD) (Pierschbacher and Ruoslahti, 1984). These peptides have dual functions: They promote cell adhesion when immobilized onto a surface, and they inhibit cell adhesion when presented to cells in solution. Adhesion proteins that contain the RGD sequence include: fibronectin, vitronectin, osteopontin, fibrinogen, von Willebrand factor, thrombospondin, laminin, entactin, tenascin, and bone sialoprotein (Ruoslahti, 1996). The RGD sequence displays specificity to about half of the 20 known integrins including the α5β1, α8β1, αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, and αiiibβ3 integrins, and, to a lesser extent, the α2β1, α3β1, α4β1, and α7β1 integrins (Ruoslahti, 1996). In particular, the αvβ3 integrin is capable of binding to a large variety of RGD containing proteins including fibronectin, fibrinogen, vitronectin, osteopontin, von Willebrand factor, and thrombospondin (Ruoslahti, 1996; Haubner et al., 1997), while the α5β1 integrin is more specific and has only been shown to bind to fibronectin (D'Souza et al., 1991).
The linear peptide sequence RGD has a much lower affinity for integrins than the proteins from which it is derived (Hautanen et al., 1989). This due to conformational specificity afforded by folded protein domains not present in linear peptides. Increased functional integrin activity has resulted from preparation of cyclic RGD motifs, alteration of the residues flanking the RGD sequence, and synthesis of small molecule mimetics (reviewed in (Ruoslahti, 1996; Haubner et al., 1997)).
The X-ray crystal structure of the 10th type III domain of fibronectin (Dickinson et al., 1994), and the NMR solution structures of the murine 9th and 10th type III fibronection domains (Copie et al., 1998) containing the RGD sequence have been solved. In these structures, the GRGDSP (SEQ ID NO: 105) amino acid sequence makes a type II β-hairpin turn that protrudes from the rest of the fibronectin structure for interaction with integrin receptors.
Short RGD peptides also have been shown to assume a type II β-turn in aqueous solution, as determined by NMR (Johnson et al., 1993). Conformation and stereochemistry about the RGD motif in the form of cyclic penta- and hexa-peptides, and disulfide-constrained peptides have been studied extensively (reviewed in (Haubner et al., 1997)). Previous approaches have shown that combinations of natural and unnatural amino acids, peptidomimetics, or disulfide bonds flanking the RGD motif have been necessary to create high affinity, biologically active β-turn structures. The recent structure of an RGD β-loop mimic bound to αvβ3 (Xiong et al., 2002) has shed some interesting light on the nature of the ligand-receptor interaction and has validated the body of work encompassing the ligand-based design strategy.
Previously, phage display technology has been used to isolate cyclic peptides specific to different integrin receptors. When a random linear hexapeptide library displayed on phage was panned with immobilized integrin, the amino acid sequence CRGDCL (SEQ ID NO: 1) was isolated (Koivunen et al., 1993). It was determined that this peptide was 10-fold more potent than linear RGD hexapeptides in inhibiting the binding of attachment of α5β1 expressing cells to fibronectin (Koivunen et al., 1993). This cyclic peptide also inhibited cell adhesion mediated by αvβ1, αvβ3, and αvβ5 integrins. In another study, phage display was used to isolate selective ligands to the α5β1 αvβ3, αvβ5, and αIIb β3 integrins from phage libraries expressing cyclic peptides (Koivunen et al., 1995). It was determined that each of the four integrins studied primarily selected RGD-containing sequences, but preferred different ring sizes and flanking residues around the RGD motif. A cyclic peptide, ACRGDGWCG (SEQ ID NO: 2), was isolated that bound with high affinity to the α5β1 integrin. In addition, the cyclic peptide ACDCRGDCFCG (SEQ ID NO: 3), which contains two disulfide bonds, was shown to be 20-fold more effective in inhibiting cell adhesion mediated by the αvβ3 and αvβ5 integrins than comparable peptides with one disulfide bond, and 200-fold more potent than linear RGD peptides.
Phage display has also been used to isolate novel integrin binding motifs from peptide libraries. The cyclic peptide CRRETAWAC (SEQ ID NO: 4) was identified from a random heptapeptide phage library with flanking cystine residues (Koivunen et al., 1994). This peptide was specific for binding to the α5β1 integrin, and not the αvβ3 and αvβ5 integrins, and was determined to have an overlapping binding site with the RGD sequence. The peptide NGRAHA (SEQ ID NO: 5) was identified by phage display libraries as well (Koivunen et al., 1993), but it was later determined that the receptor for this peptide was aminopeptidase N, and not integrins as originally thought (Pasqualini et al., 2000). A synergistic binding site on the 10th domain of fibronectin (encompassing the sequence RNS) also enhances RGD binding to the α5β1 integrin (Koivunen et al., 1994; Obara and Yoshizato, 1995). In addition, the sequence PHSRN (SEQ ID NO: 6) (from the 9th domain of fibronectin), increases α5β1 integrin binding to the RGD peptide in fibronectin (Aota et al., 1994). The sequence ACGSAGTCSPHLRRP (SEQ ID NO: 7) was identified from a 15-mer phage library panned with αvβ3 integrin. The SAGT tetrapeptide is found in the sequence of vitronectin, suggesting that this may be an accessory site for integrin recognition and binding (Healy et al., 1995). It has been hypothesized that other synergy sites may exist (reviewed in Ruoslahti, 1996), suggesting that random peptide library screening for integrin ligands other than RGD would be useful.
The presentation of multiple RGD motifs within one molecule has been shown to increase integrin binding affinity and activity. Numerous studies have demonstrated that multivalent clustering of RGD ligands within a polymer coated surface or bead results in enhanced cell adhesion, due to increased local concentration of ligand, or increased ligand/receptor avidity. (Miyamoto et al., 1995; Maheshwari et al., 2000; Pierschbacher et al., 1994; Shakesheff et al., 1998). Soluble RGD repeats incorporated into polypeptides (Saiki, 1997), or linked through a poly(carboxyethylmethacrylamide) backbone (Komazawa et al., 1993) have demonstrated an increased potential for inhibition of cancer metastasis compared to free peptide. More recently, soluble multivalent polymers of GRGD (SEQ ID NO: 8), and copolymers of GRGD and the α5β1 synergy peptide SRN have been prepared synthetically through ring-opening metathesis (Maynard et al., 2001). Homopolymers containing GRGD peptides were more potent inhibitors of fibronectin cell adhesion (IC50=0.18 mM) than peptide alone (IC50=1.08 mM). Heteropolymers containing both GRGD and SRN peptides exhibited an enhanced ability to block fibronectin adhesion with an IC50 of 0.03 mM (Maynard et al., 2001). Although multivalent homo- and hetero-oligomers of integrin peptides demonstrated increased inhibition of cell adhesion, improvements in affinity and efficacy are contemplated through the use of multivalent frameworks.
The growth of new blood vessels, termed angiogenesis, plays an important role in development, wound healing, and inflammation (Folkman and Shing, 1992). Angiogenesis has been implicated in proliferative disease states such as rheumatoid arthritis, cancer, and diabetic retinopathy, and therefore is a relevant and attractive target for therapeutic intervention. In cancer, the growth and survival of solid tumors is dependent on their ability to trigger new blood vessel formation to supply nutrients to the tumor cells (Folkman, 1992). With this new tumor vascularization comes the ability to release tumor cells into the circulation leading to metastases. One specific approach to anti-angiogenic therapy is to inhibit cell adhesion events in endothelial cells. The αvβ3 (Brooks et al., 1994) and αvβ5 integrins (Friedlander et al., 1995), and more recently the α5β1 integrin (Kim et al., 2000), have been shown to be required for angiogenesis in vascular cells. Brooks and colleagues demonstrated that the αvβ3 integrin was abundantly expressed on blood vessels, but not on dermis or epithelial cells, and expression was upregulated on vascular tissue during angiogenesis (Brooks et al., 1994). In addition, the αvβ1 integrin has been shown to be expressed on the tumor vasculature of breast, ovarian, prostate, and colon carcinomas, but not on normal adult tissues or blood vessels (Kim et al., 2000). The αvβ3 (and αvβ5) integrins are highly expressed on many tumor cells such as osteosarcomas, neuroblastomas, carcinomas of the lung, breast, prostate, and bladder, as well as glioblastomas, and invasive melanomas (reviewed in (Haubner et al., 1997). It has also been demonstrated that the expression levels of αvβ3 and αvβ5 by the vascular endothelium of neuroblastoma was associated with the aggressiveness of the tumor (Erdreich-Epstein et al., 2000).
A monoclonal anti-αvβ3 antibody (LM609) was shown to inhibit angiogenesis by fibroblast growth factor (FGF), tumor necrosis factor-a, and human melanoma fragments (Brooks et al., 1994). The humanized version of LM609, termed Vitaxin, has been shown to suppress tumor growth in animal models (Brooks et al., 1995), and target angiogenic blood vessels (Sipkins et al., 1998). Vitaxin has undergone Phase I clinical trials in humans and appears to be safe and potentially active in disease stabilization (Gutheil et al., 2000). In another study, function-blocking anti-α5β1 monoclonal antibodies were shown to inhibit cell adhesion to fibronectin, and inhibit FGF-induced angiogenesis in vivo (Kim et al., 2000). In addition, RGD peptides selective to αv (Pasqualini et al., 1997) and α5β1 integrins (Kim et al., 2000) are relevant targets for imaging and therapeutic purposes. Bacteriophage displaying an RGD peptide (CDCRGDCFC) (SEQ ID NO: 9) with high affinity to αv integrins was shown to localize to tumor blood vessels when injected into tumor-bearing mice (Ruoslahti, 2000). In other approaches, RGD containing peptides and peptidomimetics have demonstrated promise in cancer therapy by binding to overexpressed cell surface integrins and interfering with angiogenesis and tumor blood supply. Inhibition of αvβ3 and αvβ5 integrins by cyclic RGD peptides resulted in significant reduction of functional blood vessel density, and was shown to impair tumor growth and metastasis in vivo (Brooks et al., 1994; Buerkle et al., 2002). In addition, the cyclic peptide c(RGDfV) (SEQ ID NO: 10) was shown to cause αvβ3-mediated apoptosis in human malignant glioma cells (Chatterjee et al., 2000) and prostate cancer cells (Chatterjee et al., 2001). The cyclic peptide antagonist CRRETAWAC (SEQ ID NO: 11), and the nonpeptide antagonist SJ749, were shown to selectively inhibit α5β1-mediated cell adhesion to fibronectin, as well as block FGF-induced angiogenesis in vivo (Kim et al., 2000). Of particular interest, the integrin inhibitors seem to have no effect on normal vessels, and appear to function by specifically inducing apoptosis in newly budding endothelial cells during angiogenesis (Brooks et al., 1994), and interfering with the function of metalloproteinase enzymes required for cellular invasion (Brooks et al., 1996).
Radiolabeled integrin antagonists as described below are useful in tumor targeting and imaging applications. Noninvasive methods to visualize and quantify integrin expression in vivo are crucial for clinical applications of integrin antagonists (Brower, 1999). The first generation of radioiodinated cyclic RGD peptides exhibited high affinity and specificity in vitro and in vivo for αvβ3 integrins however, exhibited rapid excretion and accumulation in the liver and intestines, limiting their application (Haubner et al., 1999). Modifications of these peptides with a sugar moiety reduced their uptake in the liver, and increased their accumulation in αvβ3-expressing tumors in vivo (Haubner et al., 2001). Noninvasive imaging with an 18F-labeled version of this glycoRGD peptide by positron emission tomography demonstrated receptor-specific binding and high tumor to background ratios in vivo, suggesting suitability for αvβ3 quantification and therapy (Haubner et al., 2001). In addition, RGD peptides coupled to chelating agents could be radiolabeled with 111In, 125I, 90Y, and 177Lu, enlarging their potential for both tumor imaging and radionuclide therapy (van Hagen et al., 2000). Integrin-specific antibodies can also be useful for imaging applications. Paramagnetic liposomes coated with the anti αvβ3 integrin antibody LM609 were used for detailed imaging of rabbit carcinomas for a noninvasive means to asses growth and malignancy of tumors (Sipkins et al., 1998). The small integrin binding proteins described below would therefore be very amenable to coupling to a variety of radionuclides and chemotherapeutic agents.
Patents and Publications
Ruoslahti et al., have obtained a series of patents relating to RGD peptides. For example, U.S. Pat. No. 5,695,997, entitled “Tetrapeptide,” relates to a method of altering cell attachment activity of cells, comprising: contacting the cells with a substantially pure soluble peptide including RGDX where X is any amino acid and the peptide has cell attachment activity. The patent further includes an embodiment where X is any amino acid and the peptide has cell attachment activity and the peptide has less than about 31 amino acids.
Similarly, U.S. Pat. No. 4,792,525 relates to a substantially pure peptide including as the cell-attachment-promoting constituent the amino acid sequence Arg-Gly-Asp-R wherein R is Ser, Cys, Thr or other amino acid, said peptide having cell-attachment promoting activity, and said peptide not being a naturally occurring peptide.
U.S. Pat. No. 5,169,930, to Ruoslahti, et al., relates to a substantially pure integrin receptor characterized in that it consists of an αvβ1 subunit.
U.S. Pat. No. 5,536,814, to Ruoslahti, et al., entitled “Integrin-binding peptides,” issued Jul. 16, 1996, discloses a purified synthetic peptide consisting of certain specified amino acid sequences.
U.S. Pat. No. 5,519,005, to Ofer et al., relates to certain non-peptidic compounds comprising a guanidino and a carboxyl terminal groups with a spacer sequence of 11 atoms between them, which are effective inhibitors of cellular or molecular interactions which depend on RXD or DGR recognition, wherein X is G (gly), E (glu), Y (tyr), A (ala) or F (phe). These RXD and DGR analogues are referred to as “RXD surrogates.”
US 2005/0164300 to Artis, et al., published Jul. 28, 2005, entitled “Molecular scaffolds for kinase ligand development,” discloses molecular scaffolds that can be used to identify and develop ligands active on one or more kinases, for example, the PIM kinases, (e.g., PIM-1, PIM-2, and PIM-3).
U.S. Pat. No. 6,451,976, to Lu et al., discloses a process in which dendroaspin, a polypeptide neurotoxin analogue, is modified by recombinant DNA techniques, particularly “loop grafting,” to provide a modified polypeptide.
U.S. Pat. No. 6,962,974, to Kalluri et al., issued Nov. 8, 2005, discloses recombinantly-produced Tumstatin, comprising the NCl domain of the α3 chain of Type IV collagen, having anti-angiogenic activity, anti-angiogenic fragments of the isolated Tumstatin, multimers of the isolated Tumstatin and anti-angiogenic fragments, and polynucleotides encoding those anti-angiogenic proteins.
U.S. Pat. No. 5,766,591, to Brooks et al., relates to a method of inducing solid tumor regression comprising administering an RGD-containing integrin αvβ3 antagonist.
U.S. Pat. No. 5,880,092 to Pierschbacher et al., relates to a substantially pure compound comprising an Arg-Gly-Asp sequence stereochemically stabilized through a bridge and having a molecular weight less than about 5.4 kilodaltons.
U.S. Pat. No. 5,981,468 to Pierschbacher et al., relates to a compound having a stabilized stereochemical conformation of a cyclic RGD peptide.
Koivunen et al., “Phage Libraries Displaying Cyclic Peptides with Different Ring Sizes: Ligand Specificities of the RGD-Directed Integrins,” Bio/Technology 13:265-270 (1995) discloses selective ligands to the cell surface receptors of fibronectin (α5β1 integrin), vitronectin ((αvβ3 integrin and αvβ5 integrin and fibrinogen ((αmβ3 integrin from phage libraries expressing cyclic peptides. A mixture of libraries was used that express a series of peptides flanked by a cystine residue on each side (CX5C, CX6C, CX7C) or only on one side (CX9) of the insert.
Reiss et al., “Inhibition of platelet aggregation by grafting RGD and KGD sequences on the structural scaffold of small disulfide-rich proteins,” Platelets 17(3):153-7 (May 2006) discloses RGD and KGD containing peptide sequences with seven and 11 amino acids, respectively, which were grafted into two cystine knot microproteins, the trypsin inhibitor EETI-II and the melanocortin receptor binding domain of the human agouti-related protein AGRP, as well as into the small disintegrin obtustatin.
Wu et al., “Stepwise in vitro affinity maturation of Vitaxin, an αvβ3-specific humanized mAb,” Proc. Nat. Acad. Sci. Vol. 95, Issue 11, 6037-6042, May 26, 1998, discloses a focused mutagenesis implemented by codon-based mutagenesis applied to Vitaxin, a humanized version of the antiangiogenic antibody LM609 directed against a conformational epitope of the αvβ3 integrin complex. Wu et al., “Stepwise in vitro affinity maturation of Vitaxin, an v3-specific humanized mAb,” Proc. Nat. Acad. Sci., Vol. 95, Issue 11, 6037-6042, May 26, 1998, discloses a focused mutagenesis implemented by codon-based mutagenesis applied to Vitaxin, a humanized version of the antiangiogenic antibody LM609 directed against a conformational epitope of the αvβ3 integrin complex.